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    SinoBio Biotech provides different expression systems for your protein expression, including e.coli, yeast, insect cells and mammalian cells. For each expression systems, there are diverse strains and expression vectors for your choice. The following are some general but important parameters in choosing of proper systems for your target proteins expression.

Service Terms

For details please see the separate page for each expression systems (e.coli, yeast, insect cells and mammalian cells)
Protein Expression in E. coli
	Single batch and feed batch, high cell density fermentation     Inclusion body isolation and protein refolding     Soluble protein purification in high yield
Expression in Yeast
	Expression strain construction, positive clone selection     Development of procedures and processes for protein in secretion or non secretion     Expression strain optimization and scale up production
Expression/Mammalian cell system
	Stable cell line production, optimization of stable cell line production, isolation of conditioned media production, isolation of cell pellet, transient transfection production, conditioned media production, Cell Pellet production, and high level of protein production by using transient transfection up to a level of 40 mg/L
Expression/Baculovirus system
	Recombinant virus generation, viral plaque assays, titer determination or viral purification, high titer stock production, and optimization of baculovirus expression     Purification protein from inset cell
Purification of tagged Proteins
	One step purification with affinity resins protein with tags: His6, Flag, Fc, GST, maltose, chitin, or glucoprotein.     Remove tag with special enzymes: Thrombin, EK, TEV, Precision, or Capase3
Further purification of protein
	Chromatography: Ion exchange, size exclusion, hydrophobic interaction.     Affinity: Heparin-sepharose, hypetate, Ciba blue-agarose, red-agarose, benzimidine-agarose, camoduline-agarose resins,     Refolding protein     Concentration     Endotoxin removal

    General full service includes the following steps:

1. Choose of proper expression systems, by the customer or discussing with the customer. 2. Choose of proper expression vectors, by the customer or discussing with the customer. 3. Sequence optimization for the exact expression systems, by the customer or discussing with the customer, or a native DNA sequence is used under customers¡¯ requirement. 4. Confirm the order by the customer via email or fax. 5. The target gene will be cloned into the proper expression vector you desired. Subclone into one of the expression vector will be free if the synthesis is provided by SinoBio. 6. Sequence validation. 7. Expression screen. 8. Large scale expression and purification. 9. Quality control and confirm with the customer for the final products. 10. Shipment.   * Interim reports will send to customers for the synthesis, expression screen and purification, or a rotun reports for every two week, and all expectance will be reported to and discussed with the customer immediately.

Confidentiality
    Your confidential sequence to us by mail will be covered. We will sign non-disclosure or confidentiality agreements when necessary.
    A general template for MTA and CFA are available and you may change the terms according to your requirements and discuss it with SinoBio.

Delivery
    Materials delivered to customers include the following:
        1. Expression strain when required.
        2. DNA sequence data (When necessary.).
        3. Required final products in conventional buffer.
        4. Manufacture reports, including the major process, interim results, quality control.
Delivery time:
    From subcloning to expression, require approximately 3-12 weeks for completion. The time may vary depending on the complexity of the project.
    The typical time for shipment is about 4 business days. This may vary depending on where the customer located and accidentally, delayed by custom check.
    The final products will be shipped via fedex. DHL is optional according to the customer¡¯s requirements.

Request to the Customers

    1. Tell us the target DNA sequences or Genebank accession number or protein sequence through the E-mail.
    2. Provide us the requirements of the final products essentially the purity and amnount, other quality control requirements are optional with additional charge such as HPLC, AA Sequencing, MS, MS/MS, biological activity, endotoxin control etc. For details please read protein analysis.
    3. The customers need to pay for 50% fees as subscription for the first order.
    4. For more genes to be expressed or complex project, please contact us for a quote.

Correlative Service

Why SinoBio
    Professional: SinoBio Biotech focus on recombinant protein manufacture only, and the focus makes us more professional. All of the group leaders are come from big biopharmaceuticals with more than 10 years experience on protein production. The company are well equipped with Bioengenering fermentor, AKATA, HPLC and we can provide cGMP standard for your production. The fermentation scale is from liters to 500 liters. Final production is scale from milligram to tens of gram.
    Expertise: SinoBio's protein experts have more than 300 different protein purification experience, involved in NINE biologics development, most of them are in market and some of them in clinical trails. They also developed about 40 qualified cytokines and tens of biological enzymes.
    Quality: Diverse quality control method available for your choice such as amino acid sequencing, MS, MS/MS, LAL test, HPLC in addition to conventional SDS-PAGE analysis.
    Speed: Fast turnaround time (~10 days for a basic order)
    Price: Competitive prices as low as 800$
    Confidentiality: Strictly confidential customer's data. SinoBio will sign non-disclosure or confidentiality agreements when needed.


Why You Need Outsourcing
    Not all the protein needed are commercial available.
    Nowadays, many proteins are physically available from commercial suppliers. However, there are a significant number of proteins that exist only as digital sequences. Also, Biological and Pharmaceutical research require proteins of man-made and predicted sequences that do not exist naturally. Custom protein manufacture provides a unique approach to obtain these otherwise unavailable proteins.

CMO can provide you special protein sequence under your requirements, Point mutation, insertion and deletion, fusion to different tag,
    In addition to non-commercial available proteins, custom protein manufacture can also provide you at least the following features:
        £¦#8226; Addition of elimination of protein modification sites such as phosphorylation, glycosylation or Pegylation sites
        £¦#8226; Division of genes into modules or cassettes for protein domain swapping, insertion, deletion or duplication
        £¦#8226; Introduction of specific mutations within a particular gene module
        £¦#8226; Chimeric/fusion protein production

CMO can provide different quality protein meet your requirements.
       Lower purity to reduce your cost esp. when using as antigen.
        Higher purity to meet your special application, e.g. crystal analysis
        Even a large amount of proteins are commercial available, your request may have some special feature such as different purity requirements. If you need small amounts of these proteins, commercial suppliers may be more suitable with more insurance.


CMO can reduce your cost of money as well as time
    Protein expression and purification is still an art of skill. Not a theory can give a promised suggestion of how to get your desired protein. SinoBio Biotech have more than 300 recombinant protein manufacture experience, developed about 40 full qualified cytokines and tens of biological enzymes. Experience isn¡¯t a promise but can really improve the possibility of success.
    Weeks of time may be spent for cloning a full-length cDNA, and another couple of weeks construction and expression test, purification and quality analysis. Just thinking about the salary you have to pay for a simple protein, no matter the cost for reagents you spent.
    SinoBio Biotech, using our skilled techniques, the whole process can shorten to ONE month. If the expression strain is provide, 2-3weeks can get the aim where you want go.

CMO can reduce your adventure of fail
    For protein purification, we promised no charge if we failed to get the protein, no matter the reason is no expression or difficult to be purified. By this means, we take the adventure of a failed project, at least the time your scientist may wasted on it if it¡¯s impossible to be expressed or purified.

CMO/CRO as the first choice for your target protein:
    Large amount
    Long term usage
    No commercial available
    Special requirements such different purity
CMO/CRO as the secondary choice:
    Limit amount required, such as microgram to milligram
    Low cost product available from commercial providers
    Biological activity must be insured.

How to order
    Please download the order form to specify your information, billing information, and project information;
    Please email the completed order form to info@sinobio.net

    Please follow these instructions closely to speed up your ordering process.

    1. SinoBio Biotech adopt a non-disclosure policy to all of our customers. To protect your proprietary information please mail, email (info@sinobio.net) or fax (+8621-5079-8029) your confidentiality agreement to us for sign. If intended, this should be done before any information is released to us. If you don't have your own NDA/confidentiality agreement, you can find ours at here. Feel free to modify it to suit your specific needs.

    2. Call us (+8621-5079-8029) or email us info@sinobio.net, your gene/protein sequence and particular manipulations you wish us to perform. Particularly give clear instructions on the following options: codon optimization vs. no optimization; host organisms; single host vs. multiple host optimization. Total amount of final protein you required, the qualities and QC terms you required.



Frequently Asked Questions: choose of expression systems

    1) Should I following published papers since my aim is to use the protein as a tool but not to study the recombinant protein procedure?
    2) Should I include the signal peptide when I want to express a protein?
    3) What should I do when my target protein is predicted glycosylated?
    4) I need the target protein be phosphorylated, which expression system should I chose?
    5) How should I balance the cost, time and biological activities?
    6) Are there size limitation for recombinant protein expression?
    7) Must I optimize the codon usage for foreign gene expression? And how?
    8) I want to express my protein in the same host as the gene source, is the codon optimization necessary?
    9) When should I include a fusion tag?
    10) Will my protein be glycosylated in yeast? Is it the same to mammalian cells?
    11) What¡¯s the major protease in yeast systems? What should I care about my protein sequence?
    12) What¡¯s the difference between insect cells and mammalian cells expression system?

1) Should I following published papers since my aim is to use the protein as a tool but not to study the recombinant protein procedure?
    Enough paper work before your expression is very important because as said, no promised theory can tell which system is better. Try to copy other peoples' experience is the best way to reach your aim though it may not the best. Keep in your mind that not all of papers are trustable.

2) Should I include the signal peptide when I want to express a protein?
    Generally, signal peptide is not necessary for your target protein expression unless your purpose is to study the signal peptide itself. For E.coli expression, contains a signal peptide likely cause trouble which is apt to form inclusion bodies. Even you want the protein be secreted, e.coli, yeast and insect cells uses different signal peptide sequence and process mechanism.

3) What should I do when my target protein is predicted glycosylated?
    If native glycosylation is required, mammalian cells should be the only choice.
    Insect cells, esp mimic sf9 can mimic mammalian glycosylation in some degree and meet most of the usage of your target protein, e.g, to keep the biological activity when glycosylation is important to conserve the activity.
    Not all glycosylation is needed to keep the native biological activity of your target protein. Expression in e.coli without glycosylation is generally used for function study.

4) I need the target protein be phosphorylated, which expression system should I chose?
    Phosphorylation can occur in mammalian cells as well as insect cells and some times can also occur in bacterials, or sometimes when a kinase is co-expressed. If there¡¯s a reference for the proper phosphorylation about special expression system, try to following the paper work. Or you should test the phosphorylation by yourself. In such a case, mammalian cells should be the first choice. But you still should be aware of that, first, not all of your target protein will be phosphorylated because the signal pathway may not be activated in the particular cells you used to express your target. An alternative means is substitute the phosphorylated amino acid with phosphorylated mimic amino acid such as E for D. Some times phosphorylation of your target protein will kill the host so it¡¯s difficult to get.

5) How should I balance the cost, time and biological activities?
    If your protein only serves as antigen for antibody production, bacterial is the best choice for the time and cost. In such a case, partial of the proteins may be sufficient, such as a domain of a large protein.
    Find a balance between the cost, time and biological activity when no references can be used.When cost is the first factors to be concern, try e.coli first. Then the yeast and insect cells and the mammalian cell should be the last choice. When the biological activity is the first choice, try the opposite sequence.

6) Are there size limitation for recombinant protein expression?
    There may have no size limitation for recombinant protein. But for a particular expression system, there may have.
    For bacterial expression, generally, the size of target protein should be between 3KD-100KD. Too large protein hardly expression in e.coli and too small proteins are not stable in e.coli.
We have succeed to express 5KD-100KD protein in yeast systems.
    For insect cells and mammalian cells, the size for expression may be much broader. But too large proteins still cause problems such as degradation, low solubility, and lower expression level.

7) Must I optimize the codon usage for foreign gene expression? And how?
    Condon optimization is usually recommended to facilitate the expression. CAI index should be one of the efficient analyses in most case.

8) I want to express my protein in the same host as the gene source, is the codon optimization necessary?
    Genes from particular organism doesn¡¯t means high optimized sequence. Some of the genes use rare codons to regular the expression level, they even use some negative regulation elements inside the sequence, lacO is one of such a case.
Of course cDNAs from the same host are more possibly suitable to get high expression. After sequence analysis, if you find it¡¯s optimized, you needn¡¯t further change it.

9) When should I include a fusion tag?
    If contains a tag wont affect your application, introduce a tag should be good choice for it not only can make the purification easier, but also can facilitate the expression. Generally, TRX tag,GST tag and HIS6 tag are the most commonly used, and acceptable for most research works.

10) Will my protein be glycosylated in yeast? Is it the same to mammalian cells?
    To predict if there're some potential glycosylation site in your target protein and image if the glycosylation will affect your protein's function. Yeast will give different glycosylation to your target proteins from mammalian systems. The glycolation will not always affect the protein activities. And please note that not all potential glycosylation site will be glycosylated in yeast.

11) What¡¯s the major protease in yeast systems? What should I care about my protein sequence?
    If your target protein composed with too much basic amino acid, esp. there is several RR, RK or KR in your protein sequence, it apt to be degraded by KEX2 or YPS1. Point mutation will be recommended when such position will not affect your application. Also should be noted that not all RR site will be cut by the enzymes and not always be 100 percent digestion.

12) What¡¯s the difference between insect cells and mammalian cells expression system?
    Insect cells are more suitable for expression of large cytoplasmic proteins. The cost is much more expensive than e.coli or yeast systems. Because your target protein are most likely expressed in a soluble form, it's apt to be degraded, and the productivity is much lower compared to e.coli.
    Mammalian cells can give more promise of your target protein activities, but should be notice that the cost is the most expensive and it's difficult to scale up. If the costs of the first three are process dependent, the mammalian system is scale dependent. And the same problem as insect cells that the target protein is apt to be degraded.